Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae) Narkhyun Baea , Martin Lödlb , Arnold Pollaka , Gert Lubec a,⁎ a

Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria Naturhistorisches Museum Wien, Burgring 7, 1010 Vienna, Austria

b

AR TIC LE I N FO

ABS TR ACT

Article history:

Although several insect cuticular genes and proteins are annotated and an arthropod

Received 24 May 2011

cuticular database is available, mass spectrometrical data on cuticular proteins and their

Accepted 22 August 2011

post-translational modifications are limited.

Available online 28 August 2011

Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or

Keywords: Cuticular proteins Butterfly wings Hebemoia glaucippe Mass spectrometry Posttranslational modifications

homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

In a recent publication, structural cuticular proteins from arthropods annotation, nomenclature and sequence characteristics are reviewed [1] and an arthropod database is publicly available [2]. As to insect cuticular proteins Andersen et al. [3] have given a most useful introduction into cuticular pro-

teins (CP) and reviewed structure and function. In Lepidoptera a series of cuticular protein families are known and consist of CPR with the R&R consensus sequence, CPF/CPFL, Tweedle (TWDL), CPLCP, CPG, CPAP3, BcNCP1 orthologs, 18 aa motif, CP with less than 3 AAP and dumpy family members. Description of these families is provided in the above mentioned review and it must be stated that most informa-

⁎ Corresponding author at: Medical University of Vienna, Department of Pediatrics, Waehringer Guertel 18, A-1090 Vienna, Austria. Tel.: +43 1 404003215; fax: +43 1 404006065. E-mail address: [email protected] (G. Lubec). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.08.017

518

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 5 1 7 –53 1

tion is given at the nucleic acid/genomic level rather than at the protein level. Homology structural models of the R&R consensus region from a lepidopteran RR-1 protein and from a composite of

numerous RR-2 proteins were constructed [4,5] and presented a half barrel structure with an opening nicely sized to hold a chitin chain [1] containing an aromatic residues thought to be essential for chitin binding [6]. Bombyx mori (Linnaeus, 1758) (Fam. Bombycidae) has 89 RR-2 genes [7] and the authors propose that the large number of genes serves the possibility of divergence of scale structures to adapt to the environment in lepidopteran species [8]. Chitin-binding was also shown for the B. mori TWDL family as direct measurement of the binding of a recombinant CPT1 protein to chitin beads was observed [9]. CPLCP proteins were detected in B. mori [10] but no function could be assigned to these proteins. Several CPG genes have been identified in B. mori and are characterized by their high glycine content [11] mostly with the repeats GGYGG or GGxGG. In P. xuthus (Linnaeus, 1767) (Fam. Papilionidae) a lepidopteran specific CPG with GGY motifs was identified and indeed, the CPG family seems to be restricted to Lepidoptera. There are motifs or short stretches of amino acids that are commonly observed in CPs with unknown function [3]: The 18 aa amino acid motif was described in CPs of B. mori by Nakato et al. [12]. Finally, there are 34 proteins in Bombyx considered CPH for hypothetical cuticular proteins, because there is no evidence for participation in cuticle formation [1]. Although assignment of lepidopteran proteins into twelve CP families was shown, there is enormous variation of sequences hampering final annotation. Studies on cuticular proteins may be important for the areas of insecticide resistance [13,14], drought resistance [15] or resistance against heavy metals [16] and for butterfly aerodynamics [17] to name a few. Studying Hebemoia glaucippe wing proteins systematically, we aimed to demonstrate soluble CPs primary structure, cross-linking amino acids and post-translational modifications by a gel-based mass spectrometrical technique.

2.

Materials and methods

Focus of the investigation was the Great Orange Tip (H. glaucippe) (Fig. 1A) which belongs to the butterfly family Yellows and Whites (Fam. Pieridae). H.glaucippe is a mostly common and widespread Indo-Australian species ranging with numerous subspecies from northern India to the Maluku Islands. We used a sample of specimens from West Malaysia (Cameron Highlands region). The diversity of wing scales can be remarkable in butterflies, but the major part of the surface of H. glaucippe is covered by average, flat scales with a common basic structure. The scales are flattened and exhibit a stalk with which the scale is inserted in the wing surface. The 3d-structure is characterized by ridges of overlapping plates (r) (Fig. 1B) connected by cross-bridges (cb) (Fig. 1B). These cross-bridges Fig. 1 – (A) Hebemoia glaucippe. (B), (C) Scanning electron microscopy analysis of Hebemoia glaucippe: scales are flattened and exhibit a stalk with which the scale is inserted in the wing surface. The 3d-structure is characterized by ridges of overlapping plates (r) that are connected by cross-bridges (cb). These cross-bridges are forming windows in between (w).

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

forming windows in between (w) (Fig. 1B). The arrangement of the scales is like roof tiles throughout the wing surface (Fig. 1C). Scales are the bearer of a lot of different pigments, some of them playing a role in lepidopteran metabolism.

2.1. Sample preparation for two-dimensional gel electrophoresis 0.2 g of H. glaucippe wings (dry weight) were ground to a fine powder in liquid nitrogen and directly incubated with lysis buffer containing 25 mM Tris–HCl (pH 8.5), 2.6 M thiourea, 5 M urea and 1% 2-mercaptoethanol (2-ME) at 50 °C for 2 h. Samples were mixed by inverting the centrifuge tube repeatedly. The mixed protein sample was then filtered and centrifuged at 15,000 ×g for 10 min at 4 °C. Supernatants were pooled and centrifuged again at 15,000 ×g for 60 min at 4 °C. The final supernatant was precipitated using the 2D Cleanup Kit (BioRad) as described in the supplier's manual. Precipitated proteins were resuspended in fresh urea buffer consisting of 20 mM Tris, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 10 mM 1,4-dithioerythritol, 1 mM EDTA, 1 mM PMSF, 1 tablet Complete™ from Roche Diagnostics, Mannheim, Germany, and subsequently centrifuged at 10,000 ×g. Protein concentrations were determined using the method of Bradford [18] with BSA as the standard.

2.2.

Two-dimensional gel electrophoresis (2-DE)

The precipitated proteins (200 μL containing 300 μg proteins) were subjected to immobilized 18 cm pH 3–10 nonlinear gradient strips (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Focusing started at 200 V and the voltage was gradually increased to 8000 V at 4 V/min and kept constant for a further 3 h (approximately 150,000 V totally). Prior to the second dimensional run, strips were equilibrated twice for 15 min with gentle shaking in 10 mL of SDS equilibration buffer (50 mM Tris–HCl (pH 8.8), 6 M urea, 30% v/v glycerol, 2% w/v SDS and a trace of bromophenol blue). DTT 1% (w/v) was added at the first incubation for 15 min and 4% iodoacetamide w/v instead of DTT at the second incubation step for 15 min. The second-dimensional separation was performed on 10– 16% gradient SDS-PAGE. After protein fixation for 12 h in 50% ethanol and 10% acetic acid, gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for 8 h and excess of dye was washed out from the gels with distilled water. Molecular masses were determined by running precision protein standard markers (Bio-Rad Laboratories, Hercules, CA, USA), covering the range of 10–250 kDa. Isoelectric point values were determined as given by the supplier of the immobilized pH gradient strips.

2.3.

In-gel digestion

Gel pieces of interest were cut, put into a 1.5 mL tube and washed with 10 mM ammonium bicarbonate and 50% ACN in 10 mM ammonium bicarbonate repeatedly. Addition of ACN resulted in gel shrinking and the shrunk gel plugs were then dried in a Speedvac Concentrator 5301 (Eppendorf, Germany). Dried gel pieces were re-swollen and in-gel digested

519

with 40 ng/μL trypsin (Promega, Madison, WI, USA) in digestion buffer (consisting of 5 mM octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate, pH 7.8) and incubated overnight at 37 °C. Digestion with chymotrypsin (Roche Diagnostics), 25 ng/μL was done in 25 mM NH4HCO3 with 5 mM OGP (pH 7.8) at 30 °C for 4 h. Digestion with AspN (sequencing grade, Roche Diagnostic, Mannheim, Germany), 25 ng/μL was done in digestion buffer consisting of 25 mM NH4HCO3 and incubated overnight at 37 °C. Peptide extraction was performed with 15 μL of 1% formic acid (FA) in 5 mM OGP for 30 min, 15 μL 0.1% FA for 30 min and 15 μL 0.1% FA in 20% ACN for 30 min. The extracted peptides were pooled for nano-LC-ESI-CID/ETD-MS/MS analysis.

2.4.

Phophatase treatment

The cuticular protein CPG31 spots were cut, destained and dried as shown above. The dried spots were incubated in a solution of 0.5 mL of calf intestine alkaline phosphatase (New England Biolabs, Ipswich, MA, USA) in the presence of 100 mM ammonium bicarbonate for 1 h at 37 °C. Spots were then washed using 100 mM ammonium bicarbonate, shrunk in ACN and dried in a SpeedVac.

2.5.

Nano-LC-ESI-CID/ETD-MS/MS

The HPLC used was an Ultimate 3000 system (Dionex, Sunnyvale, CA, USA) equipped with a PepMap100 C-18 trap column (300 mm × 5 mm) and PepMap100 C-18 analytic column (75 mm × 150 mm). The gradient was (A: 0.1% FA in water, B: 0.08% FA in ACN) 4–30% B from 0 to 105 min, 80% B from 105 to 110 min, and 4% B from 110 to 125 min. An HCT ultra ETD II (Bruker Daltonics, Bremen, Germany) was used to record peptide spectra over the mass range of m/z 350–1500, and MS/MS spectra in information-dependent data acquisition over the mass range of m/z 100–2800. Repeatedly, MS spectra were recorded followed by three data-dependent CID MS/MS spectra and three ETD MS/MS spectra generated from three highest intensity precursor ions. An active exclusion of 0.4 min after two spectra was used to detect low abundant peptides. The voltage between ion spray tip and spray shield was set to 1500 V. Drying nitrogen gas was heated to 150 °C and the flow rate was 10 L/min. The collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation. Multiple charged peptides were chosen for MS/MS experiments due to their good fragmentation characteristics. MS/MS spectra were interpreted and peak lists were generated by DataAnalysis 4.0 (Bruker Daltonics). MASCOT searches were done by using the MASCOT 2.2.06 (Matrix Science, London, UK) against latest NCBI database and Lepidoptera EST data base (4,952,694 sequences; 919,616,160 residues) for protein identification repeatedly. Searching parameters were set as follows: enzyme selected as trypsin (or corresponding enzymes) with max. two missing cleavage sites, species taxonomy: limited to other metazoa, a mass tolerance of 0.2 Da for peptide tolerance, 0.2 Da for MS/MS tolerance, fixed modification of carbamidomethyl (C) and variable modification of methionine oxidation and phosphorylation (Y, T, S). Positive protein identifications were based on a significant MOWSE score. Peptides matched were

520

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 5 1 7 –53 1

Table 1 – The MASCOT results of identified spots based on search against butterfly EST database and UniProtKB database. Spot no.

Genbank gi (UniProtKB no./ protein name)

Total score/ matched peptides/M.W. (Da)/pI

Observed

Mr (expt)

Mr (calc)

Peptides (score/mass error (Da)/enzyme)

498.3331 534.7643 534.7643 544.3048 630.8401 669.4375 473.2577 709.3876 788.3870 794.3956 794.3956 1047.9327 809.7909 498.3527 544.3380 630.8607 669.4620 997.4469

994.6516 1067.5140 1067.5140 1086.5950 1259.6656 1336.8604 1416.7513 1416.7606 1574.7594 1586.7766 1586.7766 2093.8508 2426.3509 994.6908 1086.6614 1259.7068 1336.9094 1992.8792

994.5157 1067.4805 1067.4805 1086.5346 1259.5526 1336.7351 1416.6633 1416.6633 1574.7134 1586.6770 1586.6770 2093.9099 2426.1787 994.5157 1086.5346 1259.5859 1336.7351 1992.8809

922.4851 760.9300 765.6844

921.4778 1519.8454 2294.0314

921.4361 1519.7518 2294.1067

793.3442 577.3300 663.9192 663.9192 663.9226 663.9226 663.9308 705.3708

792.3369 576.3227 1325.8238 1325.8238 1325.8306 1325.8306 1325.847 2817.4541

792.3654 576.302 1325.6939 1325.6939 1325.6939 1325.6939 1325.6939 2817.3066

705.3708

2817.4541

2817.3066

863.3861 863.3861 575.9546 575.9560 575.9560 505.3475 594.6892

1724.7576 1724.7576 1724.8420 1724.8462 1724.8462 1008.6804 1781.0458

1724.743 1724.743 1724.743 1724.743 1724.743 1008.5352 1780.9119

594.6957

1781.0653

1780.9119

891.5499

1781.0852

1780.9119

891.5138

1781.013

1780.9358

663.8813 442.9380 664.4064 664.4064 674.8686 891.5178 891.5446 891.5446 594.7057 594.7057 442.9373 663.9166 663.9166 663.9247

1325.7480 1325.7922 1326.7982 1326.7982 1347.7226 1781.0210 1781.0746 1781.0746 1781.0953 1781.0953 1325.7901 1325.8186 1325.8186 1325.8348

1325.6939 1325.6939 1326.6779 1326.6779 1347.6759 1780.9319 1780.9319 1780.9319 1780.9319 1780.9067 1325.6939 1325.6939 1325.6939 1325.6939

K.EKLCYVAL.D (50/0.1359/Asp-N) Q.EMATAAASTSL.E (43/0.0336/Asp-N) Q.EMATSAASTSL.E (48/0.0336/Asp-N) G.DGVSHTVPIY.E (39/0.0605/Asp-N) H.ETVYNSIMKC.D (38/0.1131/Asp-N) \.SLYASGRTTGIVL.D (70/0.1254/Asp-N) T.ERGYSFTTTAER.E (58/0.0879/Asp-N) T.ERGYSFTTTAER.E (61/0.0973/Asp-N) Q.EMSTAAASTSLEKSY.E (65/0.0461/Asp-N) L.DFEQEMATAAASTSL.E (650/0.0996/Asp-N) L.DFEQEMSTAAASTSL.E (74/0.0996/Asp-N) L.DFEQEMSTAAASTSLEKSY.E (67/−0.0590/Asp-N) T.DYLMKILTERGYSFTTTAER.E (41/0.1721/Asp-N) K.EKLCYVAL.D (46/0.1751/Asp-N) G.DGVSHTVPIY.E (43/0.1269/Asp-N) H.ETVYNSIMKC.D (35/0.1210/Asp-N/) \.SLYASGRTTGIVL.D (70/0.1744/Asp-N/) K.DLYANTVMSGGTTMYPGIA.\ (53/−0.0016/Asp-N) 21 Y.AAPAYAAPL.A29 (0.0417/29/Chy) 98 Y.SLAEPDGSIRVVDY.T111 (0.0936/70/Chy) 98 Y.SLAEPDGSIRVVDYTADPVNGF.N119 (−0.0753/79/Chy) 112 Y.TADPVNGF.N119 (− 0.0285/31/Chy) 161 L.ARAAY.A165 (0.0208/20/Chy) 36 R.NILDTPEVAQAR.A47 (34/0.1229/Try) 36 R.NILDTPEVAQAR.A47 (45/0.1229/Try) 36 R.NILDTPEVAQAR.A47 (45/0.1367/Try) 36 R.NILDTPEVAQAR.A47 (67/0.1367/Try) 36 R.NILDTPEVAQAR.A47 (35/0.1531/Try) 48 R.AAHISALQQASKNNPNPNDDGSYDPR.W. D73 (47/0.1475/Try) 48 R.AAHISALQQASKNNPNPNDDGSYDPR.W. D73 (34/0.1475/Try) 74 R.WDNEEYWQQAEGK.W86 (21/0.0146/Try) 74 R.WDNEEYWQQAEGK.W86 (58/0.0146/Try) 74 R.WDNEEYWQQAEGK.W86 (342/0.0989/Try) 74 R.WDNEEYWQQAEGK.W86 (33/0.1031/Try) 74 R.WDNEEYWQQAEGK.W86 (38/0.1031/Try) 142 R.AAHLAALSAAK.S152 (33/0.1452/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (36/0.1339/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (30/0.1534/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (27/0.1734/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (29/0.0773/Try) R.NILDTPEVAQAR.A (83/0.1541/Try) R.NILDTPEVAQAR.A (59/0.0983/Try) R.NILDTPEVAQAR.A (40/0.1203/Try) R.NILDTPEVAEAR.A (53/0.1203/Try) R.NILDTPEVAQAR.A (41/0.0468/Try) R.LAENGAGILETPEVAAAR.A (95/0.0892/Try) R.LAQNGAGILETPEVAAAR.A (61/0.1428/Try) R.LAQDGAGILETPEVAAAR.A (128/0.1427/Try) R.LAQDGAGILETPEVAAAR.A (108/0.1634/Try) R.LAQNGAGNLETPEVAAAR.A (68/0.1885/Try) 36 R.NILDTPEVAQAR.A47 (38/0.0962/Try) 36 R.NILDTPEVAQAR.A47 (45/0.1247/Try) 36 R.NILDTPEVAQAR.A47 (71/0.1247/Try) 36 R.NILDTPEVAQAR.A47 (25/0.1409/Try)

1

gi|188286429 (P49871/actin muscle)

592/13/41,777/5.22

2

gi|188286429 (P49871/actin muscle)

229/6/41,777/5.22

3a

gi|59799332 (P83994/cuticle protein 18.6, isoform A)

211/5/18,556/6.71

4a

gi|103783172 (P42852/pupal cuticle protein)

388/17/26,415/5.95

4

gi|103783172 (P42852/pupal cuticle protein)

473/12/26,415/5.95

5a

gi|103783172 (P42852/pupal cuticle protein)

473/12/26,415/5.95

521

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

Table 1 (continued) Spot no.

Genbank gi (UniProtKB no./ protein name)

Total score/ matched peptides/M.W. (Da)/pI

Observed

Mr (expt)

Mr (calc)

663.9247 663.9375 663.9375 863.4110 505.3706 891.5043 594.6875 594.7029 891.5302 594.6875 594.7029 663.9226 594.6957 891.5499 705.3708

1325.8348 1325.8604 1325.8604 1724.8074 1008.7266 1780.994 1781.0407 1781.0869 1781.0458 1781.0407 1781.0869 1325.8306 1781.0653 1781.0852 2817.4541

1325.6939 1325.6939 1325.6939 1724.7430 1008.5352 1780.9119 1780.9119 1780.9358 1780.9319 1780.9358 1780.9358 1325.6939 1780.9319 1780.9319 2817.2702

705.3708

2817.4541

2817.2702

663.9307 891.5349 891.5349 891.5408 594.7097 841.9526 842.4598 842.4598 842.5012 842.5012 562.0102 562.0102 1035.0361

1325.8468 1781.0552 1781.0552 1781.0670 1781.1073 1681.8906 1682.9050 1682.9050 1682.9878 1682.9878 1683.0088 1683.0088 2068.0576

1325.6939 1780.9319 1780.9319 1780.9319 1780.9319 1681.8495 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 2068.0661

Peptides (score/mass error (Da)/enzyme)

6

gi|103783172 (P42852/pupal cuticle protei

238/7/26,415/5.95

7

gi|215396760 (D0VEM6/putative cuticle protein CPG31)

584/8/27,704/9.64

8

gi|40902411 (F4WXB0/zinc transporter foi) gi|205367444 (F4WXB0/zinc transporter foi) gi|222473185 (D3BV52/rhomboid family protein) gi|78231085 (D0VEM6/putative cuticle protein CPG31)

70/2/2/85,830/6.51

421.7345 428.6964

841.4544 855.3782

841.4810 855.4967

R.NILDTPEVAQAR.A47 (34/0.1409/Try) R.NILDTPEVAQAR.A47 (22/0.1665/Try) 36 R.NILDTPEVAQAR.A47 (37/0.1665/Try) 74 R.WDNEEYWQQAEGK.W86 (49/0.0664/Try) 142 R.AAHLAALSAAK.S152 (33/0.1914/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (21/0.0882/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (25/0.1288/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (30/0.1750/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (24/0.1139/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (31/0.1049/Try) 213 R.LANDGSGILDTPEVAAAR.A230 (27/0.1511/Try) R.NILDTPEVAQAR.A (74/0.1315/Try) R.LAQDGAGILETPEVAAAR.A (52/0.1807/Try) R.LAQDGAGILETPEVAAAR.A (89/0.1176) R.AAHLSALQQASHNNPNPQDDGSYDPR.W (118/0.0735/Try) R.AAHLSALQQASHNNPNPQDDGSYDPR.W (115/0.1535/Try) R.NILDTPEVAQAR.A (62/0.1529/Try) R.LAQNGAGILETPEVAAAR.A (37/0.1234/Try) R.LAQDGAGILETPEVAAAR.A (115/0.1233/Try) R.LAENGAGILETPEVAAAR.A (33/0.1352/Try) R.LAQDGAGILETPEVAAAR.A (49/0.1754/Try) R.AGQANAIQNAQALDAAR.L (113/0.0411/Try) R.AGEANAIQNAQALDAAR.L (93/0.0075//Try) R.AGQADAIQNAQALDAAR.L (147/0.0075//Try) R.AGQANAIQNAQALDAAR.L (48/0.1543//Try) R.AGQANAIQDAQALDAAR.L (155/0.1543//Try) R.AGQADAIQNAQALDAAR.L (0.1752/77//Try) R.AGQANAIQDAQALDAAR.L (71/0.1752//Try) R.AQAAAIANSAAQAQAVADTVAR.N (94/−0.0084//Try) R.VATWLPR.G (32/−0.0266/Try) R.IATWLPR.G (46/−0.1184/Try)

103/2/2/85,830/6.51

421.7742 428.8314

841.5338 855.6482

841.4810 855.4967

R.VATWLPR.G (36/0.0528/Try) R.IATWLPR.G (48/0.1516/Try)

51/1/38,442/10.08

487.2176

972.4206

972.4988

R.LELSQNNR.D (53/−0.0782/Try)

322/5/27,704/9.64

841.8700 561.9127 690.3367

1681.7254 1682.7163 2067.9883

1681.8495 1682.8084 2068.0661

690.6522

2068.9348

2069.0865

690.6636

2068.9690

2069.0501

403.6681 547.0222 748.9612

805.3216 1638.0448 2243.8618

805.4447 1638.0244 2244.0377

685.3062 403.1557 403.6461 748.9881

1368.5978 804.2968 805.2776 2243.9425

1368.6997 804.4243 805.4447 2244.0919

754.3087

2259.9043

2260.0868

685.3192

1368.6238

1368.6997

5

9

10

11

12 a

13 a

gi|103783172 (P42852/pupal cuticle protein)

gi|269913875 (D0VYP9/ glyceraldehyde-3phosphate dehydrogenase) gi|269913875 (D0VYP9/ glyceraldehyde-3phosphate dehydrogenase)

242/10/26,415/5.95

188/4/37,205/7.19

167/7/37,205/7.19

36 36

R.AGQANAIQNAQALDAAR.I (103/−0.1241/Try) R.AGQANANQNAQALDAAR.I (54/−0.0921/Try) R.AQAAAVATNAAQAQAVADTVAR.N (84/−0.0778/Try) R.AQAAAVATSAVQAQAVADTVAR.N (44/−0.1517/Try) R.AQAAAVATSAAQAQAVADTVAR.N (61/−0.0812Try) 6 R.VGINGFGR.I13 (41/−0.123/Try) 6 R.VGINGFGRIGRLVLR.A20 (27/0.0203/Try) 120 K.VIISAPSADAPMFVMGVNHEK.Y140 (20/−0.1759/Try) 202 R.GAAQNIIPASTGAAK.A216 (46/−0.1019/Try) 6 R.VGINGFGR.I13 (29/−0.1274/Try) 6 R.VGINGFGR.I13 (30/−0.1670/Try) 120 K.VIISAPSADAPMFVMGVNHEK.Y140 (26/−0.1494/Try) 120 K.VIISAPSADAPMFVMGVNHEK.Y140 (25/−0.1825/Try) 202 R.GAAQNIIPASTGAAK.A216 (62/−0.0759/Try) (continued on next page)

522

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 5 1 7 –53 1

Table 1 (continued) Spot no.

Genbank gi (UniProtKB no./ protein name)

Total score/ matched peptides/M.W. (Da)/pI

Observed

Mr (expt)

Mr (calc)

685.7732 406.1758 498.3406 498.3174 669.4399 451.6584 534.9482 582.3657 694.3354 579.7092 579.7122 890.4199 777.4847 798.9067 798.9067 532.9535 532.9535 806.2081

1369.5318 810.337 994.6666 994.6202 1336.8652 1351.9534 1601.8228 1162.7168 1386.6562 1736.1058 1736.1148 1778.8252 1552.9548 1595.7988 1595.7988 1595.8387 1595.8387 2415.6025

1369.7201 810.4058 994.5157 994.5157 1336.7351 1351.7612 1601.8777 1162.5652 1386.5609 1735.9217 1735.9217 1778.9275 1552.7733 1595.7791 1595.7791 1595.7791 1595.7791 2415.7869

642.3150 642.3150 905.0116 905.0116 983.0392 655.8319 619.4166 619.4166 936.4941

1282.6154 1282.6154 1808.0086 1808.0086 1964.0638 1964.4739 1855.228 1855.228 2806.4605

1282.5790 1282.5790 1807.8952 1807.8952 1964.0466 1964.0466 1854.8748 1854.8748 2806.5341

181/3/22,822/8.64

905.0344 928.9831

1808.0542 1855.9516

1807.9316 1855.8967

699/19/27,704/9.64

842.4689 842.4689 842.4711 562.0034 842.9432 842.9432 1035.0224

1682.9232 1682.9232 1682.9276 1682.9884 1683.8718 1683.8718 2068.0302

1682.8336 1682.8336 1682.8336 1682.8336 1683.8176 1683.8176 2068.0661

690.3912

2068.1518

2068.0661

1035.5234

2069.0322

2069.0501

690.7689

2069.2849

2069.0865

838.8567

2513.5483

2513.3580

507.8686 521.8758 842.4527 842.4689 842.4689 842.4711 562.0034 842.9432 842.9432 1035.0224

1013.7226 1041.7370 1682.8908 1682.9232 1682.9232 1682.9276 1682.9884 1683.8718 1683.8718 2068.0302

1013.5254 1041.5931 1682.8812 1682.8336 1682.8336 1682.8336 1682.8336 1683.8176 1683.8176 2068.0661

690.3912

2068.1518

2068.0661

14

gi|188246236 (P49871/actin muscle)

374/8/41,777/5.22

15 a

gi|183979394 (B2DBJ0/cuticular protein CPR59A)

306/11/22,822/8.64

25,178/22,822/8.64

16

17

18

19

gi|222465852 (B2DBJ0/Cuticular protein CPR59A) gi|103789835 (D0VEM6/putative cuticle protein CPG31)

gi|91832985 (D0VEM6/putative cuticle protein CPG31) gi|103789835 (D0VEM6/putative cuticle protein CPG31)

155/4/27,704/9.64

699/19/27,704/9.64

Peptides (score/mass error (Da)/enzyme)

202

R.GAAQNIIPASTGAAK.A216 (44/−0.1883/Try) K.ITGMAFR.V235 (22/−0.0688/Try) Q.EKLCYVAL.D (49/0.1509/Asp-N) K.EKLCYVAL.D (34/0.1045/Asp-N) \.SLYASGRTTGIVL.D (41/0.1302/Asp-N) Y.EGYALPHAILRL.D (50/0.1992/Asp-N) N.ELRVAPEEHPVLLT.E (50/−0.0549/Asp-N) F.EQEMATAAAST.\ (43/0.1516/Asp-N) L.DFEQEMSTAAAST.\ (54/0.0953/Asp-N) 47 Y.KSQVETRVGGTVKGQY.S62 (0.1841/53/Chy) 47 Y.KSQVETRVGGTVKGQY.S62 (0.1931/31/Chy) 47 Y.KSQVETRVGGTVKGQY.S62 (0.1931/48/Chy) 63 Y.SLLDADGTKRTVDY.A76 (0.1815/36/Chy) 63 Y.SLLDADGTKRTVDY.A76 (0.0197/55/Chy) 63 Y.SLLDADGTKRTVDY.A76 (0.0197/72/Chy) 63 Y.SLLDADGTKRTVDY.A76 (0.0596/74/Chy) 63 Y.SLLDADGTKRTVDY.A76 (0.0596/35/Chy) 65 L.LDADGTKRTVDYAADDVNGF.N84 (−0.1844/30/Chy) 66 L.DADGTKRTVDY.A76 (0.0365/42/Chy) 66 L.DADGTKRTVDY.A76 (0.0365/30/Chy) 54 R.VGGTVKGQYSLLDADGTK.R71 (90/0.1134/Try) 54 R.VGGTVKGQYSLLDADGTK.R71 (28/0.1134/Try) 54 R.VGGTVKGQYSLLDADGTK.R71 (36/0.0172/Try) 54 R.VGGTVKGQYSLLDADGTK.R71 (22/0.1673/Try) 73 R.TVDYAADDVNGFNAVVR.K89 (57/0.1861/Try) 73 R.TVDYAADDVNGFNAVVR.K89 (39/0.1878/Try) 90 R.KDPAVVAAPAVVATAPAVVASAPAVVSAAR.T119 (33/−0.0736/Try) R.VGGTVKGQYSLLDADGTK.R (128/0.1226/Try) R.TVDYAPDDVNGFNAVVR.K (60/0.0550/Try) 229

R.AGQANAIQNAQALDAAR.I (75/0.0897/Try) R.AGQADAIQNAQALDAAR.I (143/0.0897/Try) R.AGQANAIQDAQALDAAR.I (151/0.0941/Try) R.AGQADAIQNAQALDAAR.I (90/0.1548/Try) R.AGQANAIQNAQALDAAR.I (62/0.0543/Try) R.AGQANAIQNAQALDAAR.I (88/0.0543/Try) R.AQAAAVATNAAQAQAVADTVAR.M (145/−0.0359) R.AQAAAVATNAAQAQAVADTVAR.M (88/0.0857) R.AQAAAVATSAAQAQAVADTVAR.M (111/−0.0179) R.AQAAAVATSAVQAQAVADTVAR.M (48/0.1984/Try) R.VTEAAARAGQANAIQNAQALDAAR.I (39/0.1902/Try) R.AQAAALENAR.A (43/0.1973//Try) R.VQAVAIANTR.A (74/0.1440//Try) R.AGQAQGITNARALDAAR.V (47/0.0097/Try) R.AGQANAIQNAQALDAAR.I (75/0.0897/Try) R.AGQADAIQNAQALDAAR.I (143/0.0897/Try) R.AGQANAIQDAQALDAAR.I (151/0.0941/Try) R.AGQADAIQNAQALDAAR.I (90/0.1548/Try) R.AGQANAIQNAQALDAAR.I (62/0.0543/Try) R.AGQANAIQNAQALDAAR.I (88/0.0543/Try) R.AQAAAVATNAAQAQAVADTVAR.M (145/−0.0359/Try) R.AQAAAVATNAAQAQAVADTVAR.M (88/0.0857/Try)

523

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

Table 1 (continued) Spot no.

20

21

Genbank gi (UniProtKB no./ protein name)

gi|215397380 (D0VEM6/putative cuticle protein CPG31)

gi|215396760 (D0VEM6/putative cuticle protein CPG31)

Total score/ matched peptides/M.W. (Da)/pI

826/24/27,704/9.64

1216/35/27,704/9.64

Observed

Mr (expt)

Mr (calc)

1035.5234

2069.0322

2069.0501

690.7689

2069.2849

2069.0865

838.8567

2513.5483

2513.3580

508.2962 640.8664 427.6189 437.6242 655.9370 561.6195 841.9624 561.9823 561.9823 842.4880 842.5000 842.5075 842.5075 614.6964 690.0898

1014.5778 1279.7182 1279.8349 1309.8508 1309.8594 1681.8367 1681.9102 1682.9251 1682.9251 1682.9614 1682.9854 1683.0004 1683.0004 1841.0674 2067.2476

1014.4842 1279.6996 1279.6996 1309.6738 1309.6738 1681.8495 1681.8495 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 1840.9101 2067.1072

1035.5198

2069.0250

2069.0501

691.0070

2069.9992

2070.0703

508.3244 514.8335 640.8423 655.9050 841.4757 841.9385 842.4535 842.4620 842.4758 561.9869 561.9869 842.9383 569.3116 685.4331

1014.6342 1027.6524 1279.6700 1309.7954 1680.9368 1681.8624 1682.8924 1682.9094 1682.9370 1682.9389 1682.9389 1683.8620 1704.9130 2053.2775

1014.4842 1027.5410 1279.6996 1309.6738 1680.8907 1681.8495 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 1683.8176 1704.8655 2053.0916

1034.5424

2067.0702

2067.1072

690.0910

2067.2512

2067.1072

1035.0241

2068.0336

2068.0661

1035.4997

2068.9848

2069.0501

1035.5231

2069.0316

2069.0501

1035.5250

2069.0354

2069.0501

690.7077

2069.1013

2069.0501

691.0551

2070.1435

2070.0341

698.0656

2091.1750

2091.0821

807.7995

2420.3767

2420.2156

Peptides (score/mass error (Da)/enzyme)

R.AQAAAVATSAAQAQAVADTVAR.M (111/−0.0179/Try) R.AQAAAVATSAVQAQAVADTVAR.M (48/0.1984/Try) R.VTEAAARAGQANAIQNAQALDAAR.I (39/0.1902/Try) R.AQAAANENAR.A (50/0.0936/Try) R.AIEAARIANAAR.A (73/0.0186/Try) R.AIEAARIANAAR.A (74/0.1353/Try) R.AVEAERVANAAR.V (67/0.1770/Try) R.AVEAERVANAAR.V (77/0.1856/Try) \.AGQANAIQNAQALDAAR.L (111/−0.0129/Try) \.AGQANAIQNAQALDAAR.L (117/0.0607/Try) \.AGQANAIQDAQALDAAR.L (86/0.0915/Try) \.AGQADAIQNAQALDAAR.L (78/0.0915/Try) \.AGEANAIQNAQALDAAR.L (72/0.1279/Try) \.AGQANAIQDAQALDAAR.L (130/0.1519/Try) \.AGQANAIQNAQALDAAR.L (85/0.1669/Try) \.AGQADAIQNAQALDAAR.L (139/0.1699/Try) R.ASAAAAAEVARAVEAER.V (43/0.1573/Try) R.AQAAAIAISAAQAQAVADTVAR.N (139/0.1669/Try) R.AQAAAIANSAAQAEAVADTVAR.N (43/0.1439/Try) R.AQAAAIANSAAQAQAVADTVAR.N (40/−0.0712/Try) R.AQAAANENAR.A (43/0.1500/Try) R.ASAAAAAEVAR.A (40/0.1114/Try) R.AIEAARIANAAR.A (73/−0.0296/Try) R.AVEAERVANAAR.V (55/0.1216/Try) R.AGQAIAIQNAQALDAAR.L (103/0.0462/Try) R.AGQANAIQNAQALDAAR.L (99/0.0129/Try) R.AGQADAIQNAQALDAAR.L (124/0.0589/Try) R.AGQANAIQNAQALDAAR.L (68/0.0759/Try) R.AGQANAIQDAQALDAAR.L (124/0.1035/Try) R.AGQANAIQDAQALDAAR.L (83/0.1053/Try) R.AGQANAIQNAQALDAAR.L (89/0.1053/Try) R.AGQANAIQNAQALDAAR.L (110/0.0445/Try) R.AGQAHAIQNAQALDAAR.L (40/0.0474/Try) R.AQAAAIANSAALAQAVADTVAR.N (35/0.1859/Try) R.AQAAAIAISAAQAQAVADTVAR.N (102/−0.0370/Try) R.AQAAAIAISAAQAQAVADTVAR.N (43/0.1439/Try) R.AQAAAIANSAAQAQAVADTVAR.N (102/−0.0324/Try) R.AQAAAIANSAAQAEAVADTVAR.N (68/−0.0652/Try) R.AQAAAIADSAAQAQAVADTVAR.N (77/−0.0185/Try) R.AEAAAIANSAAQAQAVADTVAR.N (83/−0.0146/Try) R.AQAAAIADSAAQAQAVADTVAR.N (50/0.0512/Try) R.AQAAAIANSAAQAQAVADTVAR.N (55/0.1094/Try) R.AQAAAIAHSAAQAQAVADTVAR.N (48/0.0929/Try) R.VTEAAARAGQANAIQNAQALDAAR.L (45/0.1611/Try) (continued on next page)

524

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 5 1 7 –53 1

Table 1 (continued) Spot no.

22

23

Genbank gi (UniProtKB no./ protein name) gi|215396760 (D0VEM6/putative cuticle protein CPG31)

gi|215396760 (D0VEM6/putative cuticle protein CPG31)

Total score/ matched peptides/M.W. (Da)/pI

Observed

Mr (expt)

Mr (calc)

1437/50/27,704/9.64

508.3339 514.8387 640.8752 427.5929 437.5872 655.8867 841.4486 561.3158 841.9049 561.6328 561.9675 561.9720 561.9720 842.4546 842.4546 842.4643 569.3123 1034.5255

1014.6532 1027.6628 1279.7358 1279.7569 1309.7398 1309.7588 1680.8826 1680.9256 1681.7952 1681.8766 1682.8807 1682.8942 1682.8942 1682.8946 1682.8946 1682.9140 1704.9151 2067.0364

1014.4842 1027.5410 1279.6996 1279.6996 1309.6738 1309.6738 1680.8907 1680.8907 1681.8495 1681.8495 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 1704.8655 2067.1072

690.1030

2067.2872

2067.1072

1035.0113

2068.0080

2068.0661

690.4119

2068.2139

2068.0661

1035.5107

2069.0068

2069.0501

1035.5107

2069.0068

2069.0501

1035.5286

2069.0426

2069.0501

1035.5475

2069.0804

2069.0501

690.7461

2069.2165

2069.0501

690.7461

2069.2165

2069.0501

690.7571

2069.2495

2069.0501

350.7903 508.3110 514.8440 640.8600 427.5778 655.9111 841.4778 841.9463 842.4493 561.9783 561.9797 842.4673 842.4673 561.9843 561.9843 562.0069 1034.5076

699.5660 1014.6074 1027.6734 1279.7054 1279.7116 1309.8076 1680.9410 1681.8780 1682.8840 1682.9131 1682.9173 1682.9200 1682.9200 1682.9311 1682.9311 1682.9989 2067.0006

699.4028 1014.4842 1027.5410 1279.6996 1279.6996 1309.6738 1680.8907 1681.8495 1682.8336 1682.8336 1682.8084 1682.8336 1682.8336 1682.8336 1682.8336 1682.8336 2067.1072

1035.0348

2068.0550

2068.0661

1035.4684

2068.9222

2069.0501

1508/41/27,704/9.64

Peptides (score/mass error (Da)/enzyme)

R.AQAAANENAR.A (49/0.1690/Try) R.ASAAAAAEVAR.A (40/0.1218/Try) R.AIEAARIANAAR.A (75/0.0362/Try) R.AIEAARIANAAR.A (69/0.0573/Try) R.AVEAERVANAAR.V (74/0.0660/Try) R.AVEAERVANAAR.V (77/0.0850/Try) R.AGQAIAIQNAQALDAAR.L (100/−0.0080/Try) R.AGQAIAIQNAQALDAAR.L (63/0.0349/Try) R.AGQANAIQNAQALDAAR.L (138/−0.0543/Try) R.AGQANAIQNAQALDAAR.L (83/0.0270/Try) R.AGQANAIQDAQALDAAR.L (62/0.0471/Try) R.AGEANAIQNAQALDAAR.L (79/0.0606/Try) R.AGQADAIQNAQALDAAR.L (81/0.0606/Try) R.AGQANAIQNAEALDAAR.L (92/0.0611/Try) R.AGQADAIQNAQALDAAR.L (140/0.0611/Try) R.AGQANAIQDAQALDAAR.L (120/0.0805/Try) R.AGQANAIQHAQALDAAR.L (58/0.0495/Try) R.AQAAAIAISAAQAQAVADTVAR.N (95/−0.0708/Try) R.AQAAAIAISAAQAQAVADTVAR.N (38/0.1799/Try) R.AQAAAIANSAAQAQAVADTVAR.N (118/−0.0580/Try) R.AQAAAIANSAAQAQAVADTVAR.N (38/0.1478/Try) R.AQAAAIANSAAQAQAVADTVAR.N (57/−0.0432/Try) R.AQAAAIADSAAQAQAVADTVAR.N (84/−0.0433/Try) R.AQAAAIANSAAQAEAVADTVAR.N (93/−0.0074/Try) R.AQAAAIANSAAEAQAVADTVAR.N (79/0.0304/Try) R.AEAAAIANSAAQAQAVADTVAR.N (61/0.1664/Try) R.AQAAAIADSAAQAQAVADTVAR.N (55/0.1664/Try) R.AQAAAIANSAAQAEAVADTVAR.N (70/0.1994/Try) R.LANVQR.A (43/0.1633/Try) R.AQAAANENAR.A (43/0.1232/Try) R.ASAAAAAEVAR.A (35/0.1324/Try) R.AIEAARIANAAR.A (75/0.0058/Try) R.AIEAARIANAAR.A (71/0.0120/Try) R.AVEAERVANAAR.V (49/0.1338/Try) R.AGQAIAIQNAQALDAAR.L (109/0.0504/Try) R.AGQANAIQNAQALDAAR.L (52/0.0285/Try) R.AGQADAIQNAQALDAAR.L (139/0.505/Try) R.AGQANAIQNAEALDAAR.L (98/0.0795/Try) R.AGQANANQNAQALDAAR.L (63/0.1089/Try) R.AGQANAIQNAQALDAAR.L (88/0.0865/Try) R.AGQANAIQDAQALDAAR.L (126/0.0865/Try) R.AGQANAIENAQALDAAR.L (74/0.0975/Try) R.AGQANAIQDAQALDAAR.L (65/0.0975/Try) R.AGQADAIQNAQALDAAR.L (73/0.1653/Try) R.AQAAAIAISAAQAQAVADTVAR.N (93/−0.1066/Try) R.AQAAAIANSAAQAQAVADTVAR.N (114/−0.0110/Try) R.AQAAAIANSAAQAEAVADTVAR.N (87/−0.1278/Try)

525

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

Table 1 (continued) Spot no.

24 a

24

25 a

26 a

27

28

Genbank gi (UniProtKB no./ protein name)

Total score/ matched peptides/M.W. (Da)/pI

Observed

Mr (expt)

Mr (calc)

1035.5228

2069.0310

2069.0501

1035.5356

2069.0566

2069.0501

1035.5462

2069.0778

2069.0501

690.7480

2069.2222

2069.0501

698.0563

2091.1471

2091.0821

779.405

2335.193

2335.188

754.4493 754.4493 754.9102 754.9394 754.9394 503.6423 754.9679 755.4639 755.4639 504.1981 504.1981 619.7364 754.8914 503.6479

1506.884 1506.884 1507.806 1507.864 1507.864 1507.905 1507.921 1508.913 1508.913 1509.573 1509.573 1856.187 1507.7682 1507.9219

1506.827 1506.827 1507.774 1507.774 1507.774 1507.774 1507.774 1508.758 1508.758 1509.742 1509.742 1856.015 1507.7283 1507.7283

754.8732 754.8732 503.5859 754.9334 754.9334 503.9522 503.9522 751.3365

1507.732 1507.732 1507.736 1507.852 1507.852 1508.835 1508.835 2250.9877

1507.774 1507.774 1507.774 1507.774 1507.774 1508.758 1508.758 2251.1193

751.3365

2250.9877

2251.1193

754.4771 754.9122 754.9122 754.9202 527.9195 842.0314

1506.9396 1507.8098 1507.8098 1507.8258 1580.7367 1682.0482

1506.8267 1507.7743 1507.7743 1507.7743 1580.8032 1681.8495

43/1/31,454/7.64

421.7568 435.8843 718.5144

841.4990 869.7540 717.5071

55/1/210,310/5.93

755.4639

1508.9132

37/2/37,682/6.19

421.7976

841.5806

27/1/45,568/3.83

522.8932

1043.7718

gi|103777441 (B2DBJ4/putative cuticular protein)

549/13/14,602/6.02

gi|103777441 (B2DBJ4/putative cuticular protein) gi|103777441 (B2DBJ4/putative cuticular protein)

54/2/14,602/6.02

gi|103777441 (B2DBJ4/putative cuticular protein)

gi|103783201 (D0VEM6/putative cuticle protein CPG31) gi|159666375 (B4JS24/GH19024) gi|215331046 (C1MS56/predicted protein) gi|40841161 (A7ERY0/putative uncharacterized protein) gi|160487428 (A1DYI5/cathepsin B-like cysteine proteinase) gi|193742853 (C7NM68/polysaccharide deacetylase)

389/7/14,602/6.02

285/914,602/6.02

90/1/27,704/9.64

77/2/89,358/5.23

Peptides (score/mass error (Da)/enzyme)

R.AQAAAIADSAAQAQAVADTVAR.N (94/−0.0191/Try) R.AQAAAIANSAAEAQAVADTVAR.N (103/0.0066/Try) R.AEAAAIANSAAQAQAVADTVAR.N (99/0.0278/Try) R.AQAAAIADSAAQAQAVADTVAR.N (72/0.1721/Try) R.AQAAAIAHSAAQAQAVADTVAR.N (41/0.0650/Try) 48 R.AIGESQARAAEAVIQHNTEAVR.Q69 (19/0.0052//Try) 56 R.AAEAVIQHNTEAVR.Q69 (23/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (71/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (67/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (40/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (55/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (31/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (50/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (68/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (71/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (84/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (64/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (44/0.0574/Try) R.AAEAVILADTEAVR.L (37/0.0339/Try) R.AAEAVILADTEAVR.L (48/0.1963/Try) 56

R.AAEAVIQHNTEAVR.Q69 (34/0.0574/Try) R.AAEAVIQHNTEAVR.Q69 (71/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (78/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (71/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (72/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (88/0.0574/Try) 56 R.AAEAVIQHNTEAVR.Q69 (66/0.0574/Try) 48 R.AIGESQARAAEAVIQHNTEAVR.Q69 (29/−0.1316/Try) 48 R.AIGESQARAAEAVIQHNTEAVR.Q69 (52/−0.1316/Try) 56 R.AAEAVIQHNTEAVR.Q69 (78/0.113/Try) 56 R.AAEAVIQHNTEAVR.Q69 (36/0.113/Try) 56 R.AAEAVIQHNTEAVR.Q69 (36/0.0356/Try) 56 R.AAEAVIQHNTEAVR.Q69 (40/0.0516/Try) 56 R.AAEAVIQHNTEAVR.Q69 (36/−0.0666/Try) R.AGQANAIQNAQALDAAR.I (90/0.1987/Try) 56

841.53850 \.VATVIALR.R (41/−0.0395/Try) 869.56980 \.LATVIALR.R (42/0.1842/Try) 717.35920 R.CAAAAVR.R (45/0.1479/Try)

1508.7219

R.QSEPIEHTAAGDVR.R (55/0.1913/Try)

841.50210 K.VASISIPR.V (40/0.0785/Try)

1043.6491

K.WKIKTLQK.N (33/0.1227/Try)

(continued on next page)

526

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 5 1 7 –53 1

Table 1 (continued) Spot no.

29

Genbank gi (UniProtKB no./ protein name)

Total score/ matched peptides/M.W. (Da)/pI

gi|215329878 87/2/22,741.9.22 (C5XPL6/putative uncharacterized protein) gi|3738864 82/2/59,328/6.40 (F4WQS6/putative adenosylhomocysteinase 3) gi|298310691 68/2/59,326/5.66 (E1ZYV6/abhydrolase domain-containing protein 4)

Observed

Mr (expt)

740.4798 770.9054

739.4725 1539.7962

769.5101 770.9054

768.50280 768.48580 R.TLLPVAR.K (47/0.0171/Try) 1539.7962 1539.9321 R.TRSTRRTLLPVAR.K (42/−0.1359/Try)

734.4477

733.4404

Mr (calc)

Peptides (score/mass error (Da)/enzyme)

739.43410 K.GQIPGIR.P (49/0.0385/Try) 1539.9449 R.KAAPGRKGQIPGIR.P (46/-0.1486/Try)

733.4122

\.AFLNAAK.V (48/0.0282/Try)

Molecular weight and pI value of EST data base searched proteins were calculated by Compute pI/Mw tool (www.Expasy.org). Genbank gi is the matched EST identity in GenBank.Matched peptides includes the unique peptides after in situ enzyme digestion. a Identified proteins were searched on NCBI database.

then submitted to the NCBI to BLAST against UniProtKB protein database. After protein identification, an error-tolerant search was done to detect unspecific cleavage and unassigned modifications. Protein identification and modification information returned were manually inspected and filtered to obtain confirmed protein identification and modification lists. PTM searches were also done using the Modiro® software with following parameters: enzyme selected as used with two maximum missing cleavage sites, a peptide mass tolerance of 0.2 Da for peptide tolerance, 0.2 Da for fragment mass tolerance, modification 1 of carbamidomethyl (C) and modification 2 of methionine oxidation. Searches for unknown mass shifts, for amino acid substitution and calculation of significance were selected on advanced PTM explorer search strategies. A list of 172 common modifications was selected and added to virtually cleaved and fragmented peptides searched against experimentally obtained MS/MS spectra.

3.

substances are known with a repellent impact. In other groups like owl moths (Fam. Noctuidae, subfamily Catocalinae and Calpinae) long hair-like scales on the inner margin of the hindwings are reported to cause irritation of the respiratory tract of predators. Mechanical irritation is discussed as well as scale proteins causing anaphylactic reactions of the mucosa. Table 1 shows Genbank gi (UniProtKB number and protein name, total scores, matched peptides, molecular weight and pI, observed molecular weight, Mr (expected), Mr (calculated), and peptide sequences with ion scores, mass errors and the enzyme used for 29 spots (visualized by Coomassie blue staining) that were identified and the 2DE pattern is provided in Fig. 2. Putative cuticular proteins CPG31 were represented by ten spots, CPR59A was represented by two spots and both putative proteins were herein shown to exist at the protein level.

Results and discussion

As pointed out in the “Materials and methods” section the wing scales of H. glaucippe are of common structure. Yellows and Whites do not exhibit iridescent colors (except of some members of the African genus Colotis Hübner, 1819). The scales show a relatively wide distance between the ridges of overlapping plates (r in Fig. 1B). These ridges do not converge and show a more or less gill-like structure. In iridescent scales (like the neotropical genus Morpho Fabricius, 1807) we found gills on the surface of the scale with a width below 0.5 μm and not the common structure with cross-bridges and windows. The scales of Yellows and Whites commonly are well known as bearers of pteridines mainly occurring in the form of pterin. Pteridines are reported to be part of the pierid metabolism and occur mainly in white, yellow or pale red colors. Only little part of the pterin in the wings of Pieridae seems to represent stored excretory products but are synthesized at the wing site itself [19]. On the wings of Hebomoia species no

Fig. 2 – A 2DE image of Hebemoia glaucippe is shown providing assignments of identified protein spots.

527

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

Molecular weights were between approx. 14,602 and 27,704 Da and pIs are given in Tables 1 and 2. Sequence coverages of cuticular proteins were between 10% and 32.4% when the Mascot search engine was used and between 27.08% and 70.27% when Modiro searches were carried out. Using Modiro searches, sequence coverage was higher in some proteins using this search engine (Table 2). Results of Blastp searches are shown in supplemental Table 1. The unusually low sequence coverages that resulted are probably due to the specific primary structure and protein modifications of these structural proteins because the use of in-gel digestion in our laboratory using trypsin and chymotrypsin leads to much higher sequence coverages [20]. Moreover, cuticular proteins are rich in alanine, relatively hydrophobic and strongly acidic [1]. Unambiguous protein identification according to the guidelines for unambiguous protein identification [21] was achieved, however. The list of peptides identified is given in Table 1 and reveals peptide sequences in the one letter code, mass errors in daltons and lists the enzyme that generated the corresponding peptide. Protein modifications are shown in Table 3 and methylation was shown on a single peptide from cuticle protein 18.6 isoform A, revealed by CID and ETD fragmentations (supplemental Fig. 1A). Methylation has been considered a technical artifact but herein methanol was replaced by ethanol and indeed, methylation has been shown to be a possible post-translational modification [22]. Carbamylation may be considered a technical artifact. Deamidation was observed on several cuticular proteins and although it may be formed during the analytical step or storage, deamidation may well represent a biologically relevant modification: it takes place during lifetime of proteins [23,24]. Deamidation based upon misalignment was ruled out by manual inspection of spectra [25]. Phosphorylations and quinone formation may represent post translational modifications. Representative spectra are shown in supplemental Fig. 1B–D. Spot number 20

representing putative cuticle protein CPG31 showed phosphorylations on S214, spot number 23 representing the identical protein showed T213 phosphorylation only. Spot numbers 21 and 22 showed both phosphorylations at T213 and S214. Phosphatase treatment reduced the mass shifts and provides evidence for phosphorylation ruling out sulfonation with a comparable mass shift change. Spectra for phosphorylation and dephosphorylation are shown in supplemental Fig. 1. The role for phosphorylation of a cuticular protein remains elusive but may be important for mechanoelastic properties, a phenomenon shown for silk proteins [26]. Cuticle protein 18.6 has been identified in several arthropods and insects but so far not in Lepidoptera and herein we extend the presence of this protein family in H. glaucippe. The isoform A was sequenced in Locusta migratoria[27] as a component of the cuticle which contains more than 100 different structural proteins. The tetrapeptide (A-A-P-[AV]) repeats found throughout the protein are also present in many proteins constituting the protective envelope of other species and there is one chitin-binding domain. Pupal cuticle protein (P42852) has been reported at the transcriptional level and herein we show the existence at the protein level [12]. Like cuticle protein 18.6 it contains repeats of (A-A-P-[AV]) and has a wide distribution in arthropods. In Lepidoptera the presence of this protein was shown in B. mori. Two expression forms of the poorly described putative cuticle protein CPG31, were observed in the current study. As mentioned above, the glycine-rich CPG family is abundant in lepidoptera and evidence for this protein has been reported so far only at the mRNA level. The phosphorylated protein representing by four spots (S214, T213) was the only two proteins in the study and in literature – to the best of our knowledge – that were post-translationally modified by phosphorylation. So far information on PTMs of cuticular proteins is poor and was studied by Cox and Willis [28]: periodic acid Schiff stains

Table 2 – Identified cuticular proteins from Hebemoia glaucippe. Spot NO.

Protein name (UniProtKB; NCBI)

Molecular weight (Da)/calculated pI

Sequence coverage (MASCOT/MODIRO®)

3 4 5 6 15 16 7 11 18 19 20 21 22 23 24 25 26

Cuticle protein 18.6, isoform A (P83994; gi|59799332) Pupal cuticle protein (P42852; gi|103783172) Pupal cuticle protein (P42852; gi|103783172) Pupal cuticle protein (P42852; gi|103783172) Cuticular protein CPR59A (B2DBJ0; gi|183979394) Cuticular protein CPR59A (B2DBJ0; gi|183979394) Putative cuticle protein CPG31 (D0VEM6; gi|215397360) Putative cuticle protein CPG31 (D0VEM6; gi|78231085) Putative cuticle protein CPG31 (D0VEM6; gi|91832985) Putative cuticle protein CPG31 (D0VEM6; gi|103789835) Putative cuticle protein CPG31 (D0VEM6; gi|215397380) Putative cuticle protein CPG31 (D0VEM6; gi|215397360) Putative cuticle protein CPG31 (D0VEM6; gi|215397360) Putative cuticle protein CPG31 (D0VEM6; gi|215397360) Putative cuticular protein (B2DBJ4; gi|103777441) Putative cuticular protein (B2DBJ4; gi|103777441) Putative cuticular protein (B2DBJ4; gi|103777441)

18,556/6.71 26,415/5.95 26,415/5.95 26,415/5.95 22,822/8.64 22,822/8.64 27,704/9.64 27,704/9.64 27,704/9.64 27,704/9.64 27,704/9.64 27,704/9.64 27,704/9.64 27,704/9.64 14,602/6.02 14,602/6.02 14,602/6.02

19.5%/70.3% 26.4%/32% 26.4%/30% 10%/30% 32.4%/38.7% 15.6%/34.2% 13.5%/32.6% 13.5%/31.5% 28.6%/38% 21.7%/37.2% 24.1%/37.6% 29.3%/37.6% 20.7%/37.6% 30%/37.6% 25%/32% 25%/27.08% 25%/27.08%

Sequence coverage means the number of amino acids observed by the EST translated peptide amino acid length.

528

Spot no. 3

5 6 15 16

20 21

22

Protein name (acession no.) Cuticle protein 18.6 isoform A (P83994)

Putpal cuticle protein (P42852) Putpal cuticle protein (P42852) B2DBJ0 Cuticular protein (CPR59A) B2DBJ0 Cuticular protein (CPR59A)

Putative cuticle protein CPG31 (D0VEM6) Putative cuticle protein CPG31 (D0VEM6)

Putative cuticle protein CPG31 (D0VEM6)

m/z meas. [Da]

m/z theor. [Da]

Error [Da]

z

Spectra

781.97

781.907

0.0633

2

781.97

781.907

0.0633

2

863.42

863.361

0.0594

2

576

575.91

0.0905

3

619.35

619.287

0.0633

3

619.42

619.287

0.1332

3

619.42

619.287

0.1332

3

698.04

698.011

0.0291

3

698.11

698.011

0.0991

3

698.11

698.011

0.0991

3

698.1

698.011

0.0891

3

698.04

698.011

0.0291

3

Cmpd 753, + MSn (782.0), 52.1 min Cmpd 753, + MSn (782.0), 52.1 min Cmpd 698, + MSn (863.4), 38.9 min Cmpd 503, + MSn (576.0), 38.7 min Cmpd 953, + MSn (619.3), 51.4 min Cmpd 946, + MSn (619.4), 51.0 min Cmpd 946, + MSn (619.4), 51.0 min Cmpd 522, + MSn (698.0), 44.8 min Cmpd 537, + MSn (698.1), 45.0 min Cmpd 653, + MSn (698.1), 54.5 min Cmpd 475, + MSn (698.1), 44.8 min Cmpd 637, + MSn (698.0), 57.1 min

Peptide

Q1St

Score

Sig.

Y.SMeLAEMePDGSIRVVDMeY.T

55.3

288

100

Y.SMeLAEMePDGSIRVVDMeY.T

60.5

202

99

R.WDNEEYWQQAEGKCA.W

26.5

286

R.WDNEEYWQQAEGKCA.W

31

R.TVDYQuinoneAADDVNGFNAVVR.K

Modifications

Mode CID

99.9

Methylation (S98, E101, D110) Methylation (S98, E101, D110) Carbamylation (K86)

279

91.6

Carbamylation (K86)

ETD

21.8

259

99.9

Quinone (Y76)

CID

R.TVDYQuinoneAADDVNGFNAVVR.K

41

281

99.6

Quinone (Y76)

CID

R.TVDYQuinoneAADDVNGFNAVVR.K

33.6

277

97.1

Quinone (Y76)

ETD

R.AQAAAVATSHPO3AAQAQAVADAVAR.N

61.5

332

100

CID

R.AQAAAVATHPO3SAAQAQAVADAVAR.N

59.4

356

100

R.AQAAAVATSHPO3AAQAQAVADAVAR.N

49

291

100

R.AQAAAVATHPO3SAAQAQAVADAVAR.N

60.6

321

100

R.AQAAAVATSHPO3AAQAQAVADAVAR.N

54.5

270

100

Phosphorylation (S214) Phosphorylation (T213) Phosphorylation (S214) Phosphorylation (T213) Phosphorylation (S214)

ETD CID

CID CID CID CID

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 5 1 7 –53 1

Table 3 – Modifications of identified cuticular proteins on Modiro®.

23 24

25

Putative cuticular protein (B2DBJ4)

Putative cuticular protein (B2DBJ4)

698.07

698.011

0.0591

503.73

503.927

755.46

755.386

0.0736

2

856.54

856.434

0.1059

2

503.75

503.927

504.02

503.927

0.0933

3

571.49

571.292

0.1982

3

755.49

755.386

0.1036

2

504.02

503.927

0.0933

3

504.02

503.927

0.0933

3

755.42

755.386

0.0336

2

571.43

571.292

0.1382

3

−0.197

−0.177

3 3

3

Cmpd 598, +MSn (698.1), 53.9 min Cmpd 149, +MSn (503.7), 22.9 min Cmpd 154, +MSn (755.5), 23.2 min Cmpd 110, +MSn (856.5), 20.1 min Cmpd 172, +MSn (503.8), 25.0 min Cmpd 151, +MSn (504.0), 23.7 min Cmpd 176, +MSn (571.5), 20.5 min Cmpd 220, +MSn (755.5), 24.0 min Cmpd 179, +MSn (504.0), 23.6 min Cmpd 179, +MSn (504.0), 23.6 min Cmpd 180, +MSn (755.4), 23.6 min Cmpd 134, +MSn (571.4), 20.0 min

R.AQAAAVATHPO3SAAQAQAVADAVAR.N

61.9

362

R.AAEAVIQHNTEAVRDeamid.Q

54.6

375

R.AAEAVIQHNTEAVRDeamid.Q

71.5

R.AAEAVIQHNHexNAcTEAVR.Q

100

CID

99.8

Phosphorylation (T213) Deamidation (R69)

377

96.2

Deamidation (R69)

ETD

32

171

96.4

CID

R.AAEAVIQHNTEAVRDeamid.Q

54

311

99.9

N-Acetylhexosamine (N64) Deamidation (R69)

R.AAEAVIQHNTEAVRDeamid.Q

63.1

335

96.5

Deamidation (R69)

CID

R.AAEAVIQHNHexNAcTEAVR.Q

12

366

99.8

ETD

R.AAEAVIQHNTEAVRDeamid.Q

20

267

93.3

N-Acetylhexosamine (NST) Deamidation (R69)

R.AAEAVIQHNTEAVRDeamid.Q

65.6

401

99.7

Deamidation (R69)

CID

R.AAEAVIQHNTEAVRDeamid.Q

72.6

390

93.3

Deamidation (R69)

ETD

R.AAEAVIQHNTEAVRDeamid.Q

61.4

294

97.1

Deamidation (R69)

CID

R.AAEAVIQHNTHexNAcEAVR.Q

49.1

413

99.2

N-Acetylhexosamine (N64)

ETD

CID

CID

ETD J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

26

Putative cuticle protein CPG31 (D0VEM6) Putative cuticular protein (B2DBJ4)

529

530

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 5 1 7 –53 1

of gels, lectin binding to separated proteins were the techniques used in their study. Based upon the existence as a protein by mass spectrometry studies shown herein, we propose to change the name to cuticular protein CPG31. Cuticular protein CPR59A has been reported at the nucleic acid level and herein we show the presence as a protein [7]. It remains open whether quinone formation of this protein on tyrosine 76 (supplemental Fig. 2) serves a role but phenoloxidase, described in the cabbage butterfly (Pieris rapae (Linnaeus, 1758) (Fam. Pieridae) is a key enzyme in insect development leading to oxidation of tyrosine that in turn results into quinone formation [29]. Putative cuticular protein (B2DBJ4) was reported as one of camouflage-associated genes in Papilio xuthus[8] and we propose to re-name this cuticular protein as cuticular protein B2DBJ4 to the nomenclature commission, omitting the “putative” state as existence at the protein is described in this current report. This protein showed N-acetylhexosamine (supplemental Fig. 1E) as a post-translational modifications and indeed, this glycosylation is known to have regulatory roles and determines protein structure and binding to other proteins [30–32]. Knowledge on cuticular proteins and their protein modifications has been extended and the existence of some putative proteins has been proven by a gel-based mass spectrometrical approach. Supplementary materials related to this article can be found online at doi:10.1016/j.jprot.2011.08.017.

REFERENCES [1] Willis JH. Structural cuticular proteins from arthropods: annotation, nomenclature, and sequence characteristics in the genomics era. Insect Biochem Mol Biol 2010;40:189–204. [2] Magkrioti CK, Spyropoulos IC, Iconomidou VA, Willis JH, Hamodrakas SJ. cuticleDB: a relational database of Arthropod cuticular proteins. BMC Bioinformatics 2004;5:138. [3] Andersen SO, Hojrup P, Roepstorff P. Insect cuticular proteins. Insect Biochem Mol Biol 1995;25:153–76. [4] Hamodrakas SJ, Willis JH, Iconomidou VA. A structural model of the chitin-binding domain of cuticle proteins. Insect Biochem Mol Biol 2002;32:1577–83. [5] Iconomidou VA, Willis JH, Hamodrakas SJ. Unique features of the structural model of ‘hard’ cuticle proteins: implications for chitin–protein interactions and cross-linking in cuticle. Insect Biochem Mol Biol 2005;35:553–60. [6] Rebers JE, Willis JH. A conserved domain in arthropod cuticular proteins binds chitin. Insect Biochem Mol Biol 2001;31:1083–93. [7] Futahashi R, Okamoto S, Kawasaki H, Zhong YS, Iwanaga M, Mita K, et al. Genome-wide identification of cuticular protein genes in the silkworm, Bombyx mori. Insect Biochem Mol Biol 2008;38:1138–46. [8] Futahashi R, Fujiwara H. Identification of stage-specific larval camouflage associated genes in the swallowtail butterfly, Papilio xuthus. Dev Genes Evol 2008;218:491–504. [9] Tang L, Liang J, Zhan Z, Xiang Z, He N. Identification of the chitin-binding proteins from the larval proteins of silkworm, Bombyx mori. Insect Biochem Mol Biol 2010;40:228–34. [10] Cornman RS, Willis JH. Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle. Insect Mol Biol 2009;18:607–22.

[11] Zhong YS, Mita K, Shimada T, Kawasaki H. Glycine-rich protein genes, which encode a major component of the cuticle, have different developmental profiles from other cuticle protein genes in Bombyx mori. Insect Biochem Mol Biol 2006;36:99–110. [12] Nakato H, Toriyama M, Izumi S, Tomino S. Structural and expression of mRNA for a pupal cuticle protein of the silkworm, Bombyx mori. Insect Biochem 1990;20:667–78. [13] Awolola TS, Oduola OA, Strode C, Koekemoer LL, Brooke B, Ranson H. Evidence of multiple pyrethroid resistance mechanisms in the malaria vector Anopheles gambiae sensu stricto from Nigeria. Trans R Soc Trop Med Hyg 2009;103:1139–45. [14] Vontas J, David JP, Nikou D, Hemingway J, Christophides GK, Louis C, et al. Transcriptional analysis of insecticide resistance in Anopheles stephensi using cross-species microarray hybridization. Insect Mol Biol 2007;16:315–24. [15] Zhang YY, Li Y, Gao T, Zhu H, Wang DJ, Zhang HW, et al. Arabidopsis SDIR1 enhances drought tolerance in crop plants. Biosci Biotechnol Biochem 2008;72:2251–4. [16] Roelofs D, Janssens TK, Timmermans MJ, Nota B, Marien J, Bochdanovits Z, et al. Adaptive differences in gene expression associated with heavy metal tolerance in the soil arthropod Orchesella cincta. Mol Ecol 2009;18:3227–39. [17] Nachtigall W. Die aerodynamische Funktion der Schmetterlingsschuppen. Die Naturwissenschaften. Berlin/Heidelberg: Springer; 1965. p. 216–7. [18] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [19] Scoble MJ. The Lepidoptera: form, function and diversity. USA: Oxford University Press; 1993. [20] Kang SU, Fuchs K, Sieghart W, Pollak A, Csaszar E, Lubec G. Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains. Nat Protoc 2009;4:1093–102. [21] Carr S, Aebersold R, Baldwin M, Burlingame A, Clauser K, Nesvizhskii A. The need for guidelines in publication of peptide and protein identification data: Working Group on Publication Guidelines for Peptide and Protein Identification Data. Mol Cell Proteomics 2004;3:531–3. [22] Zhou W, Capello M, Fredolini C, Piemonti L, Liotta LA, Novelli F, et al. Mass spectrometry analysis of the post-translational modifications of alpha-enolase from pancreatic ductal adenocarcinoma cells. J Proteome Res 2010;9:2929–36. [23] Righetti PG. Real and imaginary artefacts in proteome analysis via two-dimensional maps. J Chromatogr B Analyt Technol Biomed Life Sci 2006;841:14–22. [24] Sarioglu H, Lottspeich F, Walk T, Jung G, Eckerskorn C. Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins. Electrophoresis 2000;21: 2209–18. [25] Mora L, Valero ML, Sanchez Del Pino MM, Sentandreu MA, Toldra F. Small peptides released from muscle glycolytic enzymes during dry-cured ham processing. J Proteomics 2011;74:442–50. [26] Winkler S, Wilson D, Kaplan DL. Controlling beta-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation. Biochemistry 2000;39: 12739–46. [27] Kalume DE, Kieffer S, Rafn K, Skou L, Andersen SO, Roepstorff P. Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman degradation and mass spectrometric techniques. Biochim Biophys Acta 2003;1645:152–63. [28] Cox DL, Willis JH. Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages. Insect Biochem 1987;17:469–84.

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 5 1 7 –5 31

[29] Xue CB, Luo WC, Jiang L, Xie XY, Xiao T, Yan L. Inhibition kinetics of cabbage butterfly (Pieris rapae L.) larvae phenoloxidase activity by 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone. Appl Biochem Biotechnol 2007;143:101–14. [30] Hagglund P, Matthiesen R, Elortza F, Hojrup P, Roepstorff P, Jensen ON, et al. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins. J Proteome Res 2007;6:3021–31.

531

[31] Kavan D, Kubickova M, Bily J, Vanek O, Hofbauerova K, Mrazek H, et al. Cooperation between subunits is essential for high-affinity binding of N-acetyl-D-hexosamines to dimeric soluble and dimeric cellular forms of human CD69. Biochemistry 2010;49:4060–7. [32] Matsuura A, Ito M, Sakaidani Y, Kondo T, Murakami K, Furukawa K, et al. O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors. J Biol Chem 2008;283:35486–95.